Anti-PD-L1 antibodies

The present application is a § 371 national phase of International Application No. PCT/CN2020/117351, filed on Sep. 24, 2020, which claims the benefit of PCT/CN2019/107689 filed on Sep. 25, 2019, all of which are entirely incorporated herein by reference.

The instant application contains a sequence listing and is hereby incorporated by reference in its entirety.

This application generally relates to antibodies. More specifically, the application relates to single-domain antibodies that specifically bind to PD-L1, a method for preparing the same, and the use thereof.

Increasing evidences from preclinical and clinical results have shown that targeting immune checkpoints is becoming the most promising approach to treat patients with cancers.

The protein Programmed Death 1 (PD-1), an inhibitory member of the immunoglobulin super-family with homology to CD28, is expressed on T cells, activated B cells, and myeloid cells (Agata et al, supra; Okazaki et al (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8) and has a critical role in regulating stimulatory and inhibitory signals in the immune system (Okazaki, Taku et al. 2007 International Immunology 19:813-824). PD-1 was discovered through screening for differential expression in apoptotic cells (Ishida et al (1992) EMBO J 11:3887-95).

PD-1 has two known ligands, PD-L1 (also named as B7-H1 or CD274) and PD-L2 (also named as B7-DC or CD273), which are cell surface expressed members of the B7 family (Freeman et al (2000) J Exp Med 192: 1027-34; Latchman et al (2001) Nat Immunol 2:261-8; Carter et al (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28 family members.

The interaction of PD-1 expressed on activated T cells, and PD-L1 expressed on tumor cells negatively regulates immune response and damps anti-tumor immunity. PD-L1 is abundant in a variety of human cancers (Dong et al (2002) Nat. Med 8:787-9). Expression of PD-L1 on tumors is correlated with reduced survival in esophageal, pancreatic and other types of cancers, highlighting this pathway as a promising target for tumor immunotherapy. Several groups have shown that the PD-1-PD-L1 interaction exacerbates disease, resulting in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1.

A single-domain antibody (sdAb) is an antibody consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. Single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446-448.); these are called VHH fragments. Currently, most research into single-domain antibodies is based on heavy chain variable domains.

Single-domain antibodies have many advantages. For instance, they generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells (Harmsen M M, De Haard H J (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77(1):13-22.). Further, they have good thermal stability and good tissue penetration. And they are also cost efficient in production. The advantages of single-domain antibodies make them suitable for various biotechnological and therapeutic applications. For instance, they are useful in the treatment of diseases, including but not limited to cancer, infectious, inflammatory and neurodegenerative diseases.

Although antibodies against PD-L1 are been developed, there are still spaces for improvement for antibody against PD-L1 as a therapeutic agent. Further, it is worth noting that there are few single-domain antibodies against PD-L1 currently. Accordingly, there is desire in the art to develop anti-PD-L1 antibodies, particularly single-domain antibodies against PD-L1.

These and other objectives are provided for by the present disclosure which, in a broad sense, is directed to compounds, methods, compositions and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present disclosure are broadly applicable in the field of antibody therapeutics and diagnostics and may be used in conjunction with antibodies that react with a variety of targets. The present disclosure provides anti-PD-L1 antibodies, preferably single-domain antibodies. It also provides methods of preparing the antibodies, nucleic acid molecules encoding the anti-PD-L1 antibodies, expression vectors and host cells used for the expression of anti-PD-L1 antibodies. The antibodies of the disclosure provide methods for treating or preventing conditions associated with PD-L1.

In some aspects, the disclosure is directed to a PD-L1 binding molecule.

In some embodiments, the PD-L1 binding molecule has one or more of the following properties:

In some embodiments, the PD-L1 binding molecule comprises at least one immunoglobulin single variable domain (for example, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1, CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, and CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3.

In some embodiments, the PD-L1 binding molecule comprises at least one immunoglobulin single variable domain (for example, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 consists of an amino acid sequence as set forth in SEQ ID NO: 1, CDR2 consists of an amino acid sequence as set forth in SEQ ID NO: 2, and CDR3 consists of an amino acid sequence as set forth in SEQ ID NO: 3.

In some embodiments, the PD-L1 binding molecule comprises at least one immunoglobulin single variable domain (for example, VHH), wherein the VHH comprises

(A) the amino acid sequence as set forth in SEQ ID NO: 4;

(B) an amino acid sequence which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 4; or

(C) an amino acid sequence with addition, deletion and/or substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids compared with SEQ ID NO: 4.

In some embodiments, the PD-L1 binding molecule is a PD-1 antagonist, for example, an anti-PD-L1 antibody.

In some embodiments, the PD-L1 binding molecule is a single-domain antibody, for example a heavy chain single-domain antibody.

In some embodiments, the PD-L1 binding molecule is a chimeric antibody or a humanized antibody.

In some embodiments, the VHH is from a camelid animal, comprising an alpaca or a llama.

In some embodiments, the VHH is fused to another molecule, comprising a Fc domain of an immunoglobin, a fluorescent protein, or a VHH with a distinct specificity.

In some embodiments, the VHH is fused to a Fc domain of IgG. In further embodiments, the PD-L1 binding molecule is a chimeric antibody of VHH from a camelid animal and Fc domain of human IgG. In further embodiments, the PD-L1 binding molecule is a chimeric antibody of VHH from a camelid animal and Fc domain of human IgG1 or IgG4.

In some embodiments, the PD-L1 binding molecule as disclosed herein is used in inhibiting or blocking the binding of PD-L1 to PD-1.

In some embodiments, the PD-L1 binding molecule as disclosed herein is used in treating or preventing a condition associated with PD-L1 in a subject.

In some aspects, the disclosure is directed to a PD-L1 binding molecule which competes for the same epitope with the PD-L1 binding molecule as disclosed herein.

In some aspects, the disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding PD-L1 binding molecule as disclosed herein.

In some embodiments, the isolated nucleic acid molecule comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 5.

In some aspects, the disclosure is directed to an expression vector comprising the nucleic acid molecule encoding the PD-L1 binding molecule as disclosed herein.

In some aspects, the disclosure is directed to a host cell comprising the expression vector as disclosed herein.

In some embodiments, the host cell is selected from, but not limited to, a bacterial cell (for example,), fungal cell (for example, a yeast) or a mammalian cell.

In some aspects, the disclosure is directed to a pharmaceutical composition comprising at least one PD-L1 binding molecule as disclosed herein and a pharmaceutically acceptable carrier.

In some aspects, the disclosure is directed to a method for preparing the PD-L1 binding molecule which comprises culturing the host cell comprising the expression vector as defined above under a condition of expressing the PD-L1 binding molecule in the host cell and isolating the PD-L1 binding molecule from the host cell.

In some aspects, the disclosure is directed to a method for inhibiting or blocking the binding of PD-L1 to PD-1 in a subject, comprising: administering a therapeutically effective amount of the PD-L1 binding molecule as disclosed herein to the subject.

In some aspects, the disclosure is directed to a method of treating a condition associated with PD-L1 in a subject, comprising: administering a therapeutically effective amount of the PD-L1 binding molecule as disclosed herein to the subject.

In some embodiments, the subject has been identified as having a disorder or a condition likely to respond to a PD-L1 antagonist.

In some aspects, the disclosure is directed to a method of treating a condition in a subject that would benefit from upregulation of immune response, comprising administering a therapeutically effective amount of the PD-L1 binding molecule as disclosed herein to the subject.

In some embodiments, the subject has upregulated expression of PD-L1.

In some aspects, the disclosure is directed to the use of the PD-L1 binding molecule as disclosed herein in the manufacture of a medicament for treating or preventing proliferative disorders such as cancers.

In some aspects, the disclosure is directed to the use of PD-L1 binding molecule as disclosed herein in the manufacture of a medicament for treating or preventing a condition that would benefit from upregulation of immune response.

In some embodiments, the condition is a proliferative disorder comprising cancer, or an infection.

In some embodiments, the cancer is selected from the group consisting of: breast, lung, colon, ovarian, melanoma, bladder, kidney, liver, salivary, stomach, gliomas, thyroid, thymic, epithelial, head and neck cancers, gastric and pancreatic cancer.

In some embodiments, the infection is a chronic infection.

In some aspects, the disclosure is directed to kits or devices and associated methods that employ the PD-L1 binding molecule as disclosed herein, and pharmaceutical compositions as disclosed herein, which are useful for the treatment of proliferative disorders such as cancer. To this end, the present disclosure preferably provides an article of manufacture useful for treating such disorders comprising a receptacle containing the PD-L1 binding molecule as disclosed herein and instructional materials for using the PD-L1 binding molecule as disclosed herein to treat, ameliorate or prevent a proliferative disorder or progression or recurrence thereof. In selected embodiments, the devices and associated methods will comprise the step of contacting at least one circulating tumor cell with the PD-L1 binding molecule as disclosed herein.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Further, the contents of all references, patents and published patent applications cited throughout this application are incorporated herein in entirety by reference.

While the present disclosure may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprising,” as well as other forms, such as “comprises” and “comprised”, is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6ed., W.B. Saunders Company (2010); Sambrook J. & Russell D.3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al.,, Wiley, John & Sons, Inc. (2002); Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al.,, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.